Fig. 6

Tpr Stabilizes Replication-dependent H1 Variant H1.1 and H1.2 by Preventing Protein Degradation. a–b mRNA levels of H1.1, H1.2 or H1x were quantified by quantitative Real-Time PCR in untransfected U2OS Tet/On cells (a) or U2OS Tet/On cells expressing H1 variants H1.1, H1.2 or H1x (b) treated with either control siRNA or siRNA targeting Tpr for 48 h. Expression levels of histone H1 genes were normalized relative to levels of GAPDH. Each experiment was completed on three biological replicates where each biological replicate was obtained in triplicate, and the mean of these values was used for further analysis. Statistical analysis was carried out using unpaired Student’s t-test: p ≤ 0.05 (*), p ≤ 0.01 (**), and p ≤ 0.001 (***). The data are presented as means ± standard deviations (n = 3). c Untransfected (UN) U2OS Tet/On cells or U2OS Tet/On cells expressing H1 variants (H1.1, H1.2 or H1x) were treated with either control siRNA or siRNA targeting protein Tpr for 72 h. For the last 3 h of siRNA transfection, 20 μM of MG132 was added to the media. 20 μg whole cell extracts of each sample were resolved on SDS-PAGE gel, and visualized by immunoblotting with antibodies targeting proteins indicated on the right. d Untransfected (UN) U2OS Tet/On cells or U2OS Tet/On cells expressing H1 variants (H1.1, H1.2 or H1x) were treated with either control siRNA or siRNA targeting protein Tpr for 72 h. For the last 3 h of siRNA transfection, 20 μM of MG132 was added to the media. Treated cells were then simultaneously stained with DAPI (blue), Tpr (Red), and 6 × His-tag (Green). Overlay of anti-Tpr and anti-His staining was shown for comparison. e Scatter plot of quantified fluorescence intensities from Fig. 6d. Quantification and regression analysis was performed as described in Experimental Procedures